RESUMEN
Human embryonic stem cells (hESCs) are self-renewing and pluripotent cells that originate from the inner cell mass of the blastocyst. Mitosis is fundamental to organism survival and reproduction and is responsible for the equal distribution of duplicated chromosomes into daughter cells. Mitotic dysfunction is associated with a wide variety of human diseases, not least cancer. hESCs have a unique cell cycle distribution, but it is unclear exactly how the mitotic activity of hESCs is related to their proliferation and differentiation. Here, we established a cell line of hESCs stably expressing GFP-α-tubulin and mCherry-H2B by lentiviral infection to analyze and visualize mitosis in detail. During metaphase, the mitotic spindle was smaller and wider and contained a greater proportion of astral microtubules than normal cells. In addition, spindle microtubules were more stable, and chromosome alignment was faster in hESCs than in somatic cells. We also found that the spindle assembly checkpoint was functional in hESCs. These findings thus reveal a specialized mitotic behavior of hESCs.
Asunto(s)
Células Madre Embrionarias Humanas/inmunología , Mitosis/inmunología , Células HeLa , HumanosRESUMEN
Although surgical interventions have become optional for refractory vitiligo, grafting related injuries is inevitable. Embryonic stem cell (ESC) derivatives can be used in transplantation to address this issue, but the immune rejection due to allogeneic transplantation is of great concern. To investigate the immunogenicity of ESC derived melanocytes (ES-MC), we established a co-culture system of ES-MC and allogeneic PBMC. The results showed that ES-MC were similar to human primary melanocytes, with low expression of immune related molecules, and limited capability of stimulating allogeneic lymphocytes in vitro. Taken together, our findings confirm that ES-MC are of limited immunogenicity, providing new insights into the application of ES-MC in the regenerative medicine such as treating vitiligo.
Asunto(s)
Células Madre Embrionarias Humanas/inmunología , Melanocitos/inmunología , Diferenciación Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Células Madre Embrionarias Humanas/citología , Humanos , Melanocitos/citología , Medicina RegenerativaRESUMEN
Antagonism of ROS signaling can inhibit cell apoptosis and autophagy, thus favoring the maintenance and expansion of hematopoietic stem cells. Alpha lipoic acid (ALA), a small antioxidant molecule, affects cell apoptosis by lowering the ROS level. In this study, we show that ALA promoted production of human pluripotent stem cells (hPSCs) derived hemogenic endothelial cells and hematopoietic stem/progenitor cells in vitro. Transcriptome analysis of hPSCs derived hemogenic endothelial cells showed that ALA promoted endothelial-to-hematopoietic transition by up-regulating RUNX1, GFI1, GFI1B, MEIS2, and HIF1A and down-regulating SOX17, TGFB1, TGFB2, TGFB3, TGFBR1, and TGFBR2. ALA also up-regulated sensor genes of ROS signals, including HIF1A, FOXO1, FOXO3, ATM, PETEN, SIRT1, and SIRT3, during the process of hPSCs derived hemogenic endothelial cells generation. However, in more mature hPSC-derived hematopoietic stem/progenitor cells, ALA reduced ROS levels and inhibited apoptosis. In particular, ALA enhanced development of hPSCs derived hematopoietic stem/progenitor cells by up-regulating HIF1A in response to a hypoxic environment. Furthermore, addition of ALA in ex vivo culture greatly improved the maintenance of functional cord blood HSCs by in vivo transplantation assay. Our findings support the conjecture that ALA plays an important role in efficient regeneration of hematopoietic stem/progenitor cells from hPSCs and maintenance of functional HSCs, providing insight into understanding of regeneration of early hematopoiesis for engineering clinically useful hPSCs derived hematopoietic stem/progenitor cells transplantation. Thus, ALA can be used in the study of hPSCs derived HSCs.
Asunto(s)
Células Madre Hematopoyéticas/inmunología , Células Madre Embrionarias Humanas/inmunología , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Ácido Tióctico/farmacología , Antígenos de Diferenciación/inmunología , Línea Celular , Células Madre Hematopoyéticas/citología , Células Madre Embrionarias Humanas/citología , Humanos , Especies Reactivas de Oxígeno/inmunología , Transducción de Señal/inmunologíaRESUMEN
Lung injury and fibrosis represent the most significant outcomes of severe and acute lung disorders, including COVID-19. However, there are still no effective drugs to treat lung injury and fibrosis. In this study, we report the generation of clinical-grade human embryonic stem cells (hESCs)-derived immunity- and matrix-regulatory cells (IMRCs) produced under good manufacturing practice requirements, that can treat lung injury and fibrosis in vivo. We generate IMRCs by sequentially differentiating hESCs with serum-free reagents. IMRCs possess a unique gene expression profile distinct from that of umbilical cord mesenchymal stem cells (UCMSCs), such as higher expression levels of proliferative, immunomodulatory and anti-fibrotic genes. Moreover, intravenous delivery of IMRCs inhibits both pulmonary inflammation and fibrosis in mouse models of lung injury, and significantly improves the survival rate of the recipient mice in a dose-dependent manner, likely through paracrine regulatory mechanisms. IMRCs are superior to both primary UCMSCs and the FDA-approved drug pirfenidone, with an excellent efficacy and safety profile in mice and monkeys. In light of public health crises involving pneumonia, acute lung injury and acute respiratory distress syndrome, our findings suggest that IMRCs are ready for clinical trials on lung disorders.
Asunto(s)
Células Madre Embrionarias Humanas/inmunología , Lesión Pulmonar/terapia , Pulmón/patología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/inmunología , Animales , Células Cultivadas , Femenino , Fibrosis , Haplorrinos , Células Madre Embrionarias Humanas/citología , Humanos , Inmunidad , Inmunomodulación , Pulmón/inmunología , Lesión Pulmonar/inmunología , Lesión Pulmonar/patología , Masculino , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos C57BLRESUMEN
In this study we used two different techniques in order to isolate pericytes from the wall of human umbilical cord vein and get two different groups of cells were named as "pellet and primer cells". These groups were compared with each other according to their morphologies and stem cell marker expressions. Also, these two different populations were compared with each other and with human bone marrow mesenchymal stem cells (BM-MSCs) according to their transcriptomic profiles. Then, pellet cells proteomic profiles were determined. Our results showed that morphologies and cell surface marker expressions of pellet cells and primer cells are similar. On the other hand, according to immunofluorescence staining results, in contrast to primer cells, pellet cells showed positive NG2 and PDGFR-ß staining. As a result of gene expression profiling, pellet cells have upregulated genes related with muscle, neural and immune cell differentiation, development and pluripotency. On the other hand, primer cells have upregulated adhesion pathway-related genes. In addition to differences between pellet and primer cells, the gene expression profiles of these cell groups are also different from BM-MSCs. The results of transcriptome and proteome analysis of pellet cells were in consistent with each other.
Asunto(s)
Células Madre Embrionarias Humanas/metabolismo , Pericitos/citología , Venas Umbilicales/citología , Adulto , Células de la Médula Ósea/citología , Antígeno CD146/biosíntesis , Antígeno CD146/inmunología , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Femenino , Sangre Fetal/citología , Sangre Fetal/inmunología , Sangre Fetal/metabolismo , Expresión Génica , Perfilación de la Expresión Génica/métodos , Células Madre Embrionarias Humanas/inmunología , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Pericitos/inmunología , Pericitos/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Células Madre/metabolismo , Transcriptoma , Cordón Umbilical/citología , Venas Umbilicales/metabolismoRESUMEN
Natural killer (NK) cells are important in the immune defense against tumor cells and pathogens, and they regulate other immune cells by cytokine secretion. Although murine NK cell biology has been extensively studied, knowledge about transcriptional circuitries controlling human NK cell development and maturation is limited. By generating ETS1-deficient human embryonic stem cells and by expressing the dominant-negative ETS1 p27 isoform in cord blood hematopoietic progenitor cells, we show that the transcription factor ETS1 is critically required for human NK cell differentiation. Genome-wide transcriptome analysis determined by RNA-sequencing combined with chromatin immunoprecipitation-sequencing analysis reveals that human ETS1 directly induces expression of key transcription factors that control NK cell differentiation (ie, E4BP4, TXNIP, TBET, GATA3, HOBIT, BLIMP1). In addition, ETS1 regulates expression of genes involved in apoptosis and NK cell activation. Our study provides important molecular insights into the role of ETS1 as an important regulator of human NK cell development and terminal differentiation.
Asunto(s)
Diferenciación Celular/inmunología , Regulación de la Expresión Génica/inmunología , Células Madre Embrionarias Humanas/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Proteína Proto-Oncogénica c-ets-1/inmunología , Apoptosis/genética , Apoptosis/inmunología , Diferenciación Celular/genética , Línea Celular , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Células Madre Embrionarias Humanas/citología , Humanos , Células Asesinas Naturales/citología , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Proteína Proto-Oncogénica c-ets-1/genéticaRESUMEN
Spinal cord injury (SCI) has been associated with a dismal prognosis-recovery is not expected, and the most standard interventions have been temporizing measures that do little to mitigate the extent of damage. While advances in surgical and medical techniques have certainly improved this outlook, limitations in functional recovery continue to impede clinically significant improvements. These limitations are dependent on evolving immunological mechanisms that shape the cellular environment at the site of SCI. In this review, we examine these mechanisms, identify relevant cellular components, and discuss emerging treatments in stem cell grafts and adjuvant immunosuppressants that target these pathways. As the field advances, we expect that stem cell grafts and these adjuvant treatments will significantly shift therapeutic approaches to acute SCI with the potential for more promising outcomes.
Asunto(s)
Rechazo de Injerto/prevención & control , Enfermedad Injerto contra Huésped/prevención & control , Inmunosupresores/uso terapéutico , Células Madre Pluripotentes Inducidas/trasplante , Células Precursoras de Oligodendrocitos/trasplante , Traumatismos de la Médula Espinal/terapia , Adyuvantes Inmunológicos , Aloinjertos , Animales , Basiliximab/uso terapéutico , Células Cultivadas , Ensayos Clínicos como Asunto , Ciclosporina/uso terapéutico , Femenino , Supervivencia de Injerto/inmunología , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/inmunología , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Masculino , Ratones , Ácido Micofenólico/uso terapéutico , Células Precursoras de Oligodendrocitos/inmunología , Ratas , Tacrolimus/uso terapéutico , Trasplante AutólogoRESUMEN
Age-related macular degeneration is caused by dysfunction and loss of retinal pigment epithelium (RPE) cells, and their transplantation may rescue visual functions and delay disease progression. Human embryonic stem cells (hESCs) may be an unlimited source of RPE cells for allotransplantation. We analyzed the immunomodulatory properties of hESC-derived RPE (hESC-RPE) cells, and showed that they inhibited T cell responses. Co-culture experiments showed that RPE cells inhibited interfon-γ secretion and proliferation of activated T cells. Furthermore, hESC-RPE cells enhanced T cell apoptosis and secretion of the anti-inflammatory cytokine interleukin-10 (IL-10). In addition, RPE cells altered the expression of T cell activation markers, CD69 and CD25. RPE cells transplanted into RCS rats without immunosuppression survived, provided retinal rescue, and enhanced IL-10 blood levels. Our data suggest that hESC-RPE cells have immunosuppressive properties. Further studies will determine if these properties are sufficient to alleviate the need for immunosuppression therapy after their clinical allotransplantation.
Asunto(s)
Células Madre Embrionarias Humanas/inmunología , Epitelio Pigmentado de la Retina/inmunología , Linfocitos T/inmunología , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Línea Celular , Técnicas de Cocultivo , Células Madre Embrionarias Humanas/citología , Humanos , Inmunomodulación , Interferón gamma/inmunología , Interleucina-10/inmunología , Lectinas Tipo C/inmunología , Activación de Linfocitos , Epitelio Pigmentado de la Retina/citología , Linfocitos T/citologíaRESUMEN
Monoclonal antibodies (mAbs) are used as targeted therapies against cancers. These mAbs kill cancer cells via various mechanisms of actions. In this study, human embryonic stem cells (hESCs) was used as the immunogen to generate a panel of antibodies. From this panel of mAbs, A19 was found to bind both hESC and various cancer cell lines. The antigen target of A19 was identified as Erbb-2 and glycan analysis showed that A19 binds to a N-glycan epitope on the antigen. A19 was elucidated to internalize into cancer cells following binding to Erbb-2 and hence developed as an antibody-drug conjugate (ADC). Using ADC as the mechanism of action, A19 was able to kill cancer cells in vitro and delayed the onset of tumour formation in mice xenograft model. When compared to Herceptin, A19 binds to different isoforms of Erbb-2 and does not compete with Herceptin for the same epitope. Hence, A19 has the potential to be developed as an alternative targeted therapeutic agent for cancers expressing Erbb-2.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino , Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/farmacología , Células Madre Embrionarias Humanas/inmunología , Neoplasias Experimentales , Animales , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Monoclonales de Origen Murino/farmacología , Antineoplásicos Inmunológicos/inmunología , Línea Celular Tumoral , Femenino , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite, several lines of evidence suggesting the possible role of hypoxia in stem cell development and differentiation its significance in conferring the stemness and pluripotency remains elusive. In the present study we sought to delineate the candidate genes and molecular pathways imposed during hypoxic microenvironment and its physiological relevance in tipping the balance between the niche and cellular differentiation. Integrated meta-analysis was performed between the hypoxia exposed and normal human embryonic stem cells, employing three transcriptomic cohorts (GSE35819, GSE9510 and GSE37761) retrieved from Gene expression omnibus (GEO) database. Results reveal that a total number of 12 genes were consistently differentially expressed (6up regulated and 6 down regulated) with FDR <0.05 and fold change >1.5. The Gene Ontology (GO) functions and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis was performed using DAVID. The GO analysis showed DEG significantly enriched in terms of Cellular process (GO:0009987), protein binding (GO:0005515) and cell part (GO:0044464). KEGG analysis indicated participation of genes associated with circadian rthyum regulation and PPAR signalling pathway. Further, gene-set signature (MsigDB) enrichment analysis showed positive regulation with inflammatory signals and negative association with PPAR and p53 pathway. Protein-protein network of gene modules suggests significant hub proteins viz. CTTNB1 (Degreeâ¯=â¯18), IL8 (Degreeâ¯=â¯15), NFKB1 (Degreeâ¯=â¯15) and RELA (Degreeâ¯=â¯15) in the PPI network. MCODE algorithm was used for subnetworks of the PPI network. Our integrative analysis documents the potential candidate genes which serves distinct roles influencing metabolic shift and induce inflammatory effectors contributing to hypoxic mediated stem cell niche.
Asunto(s)
Células Madre Embrionarias Humanas/metabolismo , Hipoxia/genética , Inflamación/genética , Regulación hacia Abajo , Ontología de Genes , Células Madre Embrionarias Humanas/inmunología , Humanos , Hipoxia/inmunología , Inflamación/inmunología , Mapas de Interacción de Proteínas , Transcriptoma , Regulación hacia ArribaAsunto(s)
Células Madre Embrionarias Humanas , Investigación con Células Madre , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Animales , Diferenciación Celular/genética , Reprogramación Celular , Ensayos Clínicos como Asunto , Diabetes Mellitus/patología , Diabetes Mellitus/terapia , Edición Génica , Glucosa/metabolismo , Haplorrinos , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/inmunología , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/trasplante , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Degeneración Macular/patología , Degeneración Macular/terapia , Ratones , Técnicas de Transferencia Nuclear , Organoides/citología , Organoides/metabolismo , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/terapia , Medicina Regenerativa/tendenciasRESUMEN
BACKGROUND & AIMS: The 4 genotypes of hepatitis E virus (HEV) that infect humans (genotypes 1-4) vary in geographical distribution, transmission, and pathogenesis. Little is known about the properties of HEV or its hosts that contribute to these variations. Primary isolates grow poorly in cell culture; most studies have relied on variants adapted to cancer cell lines, which likely alter virus biology. We investigated the infection and replication of primary isolates of HEV in hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells. METHODS: Using a cell culture-adapted genotype 3 strain and primary isolates of genotypes 1 to 4, we compared viral replication kinetics, sensitivity to drugs, and ability of HEV to activate the innate immune response. We studied HLCs using quantitative reverse-transcriptase polymerase chain reaction and immunofluorescence assay and enzyme-linked immunosorbent assays. We used an embryonic stem cell line that can be induced to express the CRISPR-Cas9 machinery to disrupt the peptidylprolyl isomerase A gene, encoding cyclophilin A (CYPA), a protein reported to inhibit replication of cell culture-adapted HEV. We further modified this line to rescue expression of CYPA before terminal differentiation to HLCs and performed HEV infection studies. RESULTS: HLCs were permissive for infection by nonadapted, primary isolates of HEV genotypes 1 to 4. HEV infection of HLCs induced a replication-dependent type III interferon response. Replication of primary HEV isolates, unlike the cell culture-adapted strain, was not affected by disruption of the peptidylprolyl isomerase A gene or exposure to the CYPA inhibitor cyclosporine A. CONCLUSIONS: Cell culture adaptations alter the replicative capacities of HEV. HLCs offer an improved, physiologically relevant, and genetically tractable system for studying the replication of primary HEV isolates. HLCs could provide a model to aid development of HEV drugs and a system to guide personalized regimens, especially for patients with chronic hepatitis E who have developed resistance to ribavirin.
Asunto(s)
Virus de la Hepatitis E/crecimiento & desarrollo , Hepatocitos/virología , Células Madre Embrionarias Humanas/virología , Células Madre Pluripotentes Inducidas/virología , Replicación Viral , Antivirales/farmacología , Diferenciación Celular , Ciclofilina A/genética , Ciclofilina A/metabolismo , Farmacorresistencia Viral , Genotipo , Células Hep G2 , Virus de la Hepatitis E/efectos de los fármacos , Virus de la Hepatitis E/genética , Virus de la Hepatitis E/inmunología , Hepatocitos/inmunología , Hepatocitos/metabolismo , Interacciones Huésped-Patógeno , Células Madre Embrionarias Humanas/inmunología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Inmunidad Innata , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/metabolismo , Cinética , Fenotipo , ARN Viral/genética , Sofosbuvir/farmacología , Factores de Tiempo , Transfección , Replicación Viral/efectos de los fármacosRESUMEN
Antibody-mediated cell killing has significantly facilitated the elimination of undesired cells in therapeutic applications. Besides the well-known Fc-dependent mechanisms, pathways of antibody-induced apoptosis were also extensively studied. However, with fewer studies reporting the ability of antibodies to evoke an alternative form of programmed cell death, oncosis, the molecular mechanism of antibody-mediated oncosis remains underinvestigated. In this study, a monoclonal antibody (mAb), TAG-A1 (A1), was generated to selectively kill residual undifferentiated human embryonic stem cells (hESC) so as to prevent teratoma formation upon transplantation of hESC-derived products. We revealed that A1 induces hESC death via oncosis. Aided with high-resolution scanning electron microscopy (SEM), we uncovered nanoscale morphological changes in A1-induced hESC oncosis, as well as A1 distribution on hESC surface. A1 induces hESC oncosis via binding-initiated signaling cascade, most likely by ligating receptors on surface microvilli. The ability to evoke excess reactive oxygen species (ROS) production via the Nox2 isoform of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is critical in the cell death pathway. Excess ROS production occurs downstream of microvilli degradation and homotypic adhesion, but upstream of actin reorganization, plasma membrane damage and mitochondrial membrane permeabilization. To our knowledge, this is the first mechanistic model of mAb-induced oncosis on hESC revealing a previously unrecognized role for NAPDH oxidase-derived ROS in mediating oncotic hESC death. These findings in the cell death pathway may potentially be exploited to improve the efficiency of A1 in eliminating undifferentiated hESC and to provide insights into the study of other mAb-induced cell death.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Apoptosis , Especies Reactivas de Oxígeno/metabolismo , Actinas/metabolismo , Secuencia de Carbohidratos , Línea Celular , Membrana Celular/metabolismo , Epítopos/inmunología , Células Madre Embrionarias Humanas/inmunología , Células Madre Embrionarias Humanas/ultraestructura , Humanos , Microscopía Electrónica de Rastreo , Membranas Mitocondriales/metabolismo , NADPH Oxidasa 2/metabolismo , PermeabilidadRESUMEN
Human embryonic stem cells (hESCs) hold great promise in the regenerative therapy of many currently untreatable human diseases. One of the key bottlenecks is the immune rejection of hESC-derived allografts by the recipient. To overcome this challenge, we have established new approaches to induce immune protection of hESC-derived allografts through the coexpression of immune suppressive molecules CTLA4-Ig and PD-L1. However, this in turn raises a safety concern of cancer risk because these hESC-derived cells can evade immune surveillance. To address this safety concern, we developed a safety checkpoint so that the immune evasive hESC-derived cells in the graft can be effectively eliminated if any cellular transformation is detected. In this context, we knock-in the suicidal gene herpes simplex virus thymidine kinase (HSVTK) into the constitutive HPRT locus of CP hESCs (knock-in hESCs expressing CTLA4-Ig and PD-L1), denoted CPTK hESCs. Employing humanized mice (Hu-mice) reconstituted with human immune system, we demonstrated that the CPTK hESC-derived cells are protected from immune rejection. In addition, CPTK hESC-derived cells can be efficiently eliminated in vitro and in vivo with FDA approved TK-targeting drug ganciclovir. Therefore, this new safety checkpoint improves the feasibility to use the immune evasive hESC-derived cells for regenerative medicine. Stem Cells 2017;35:1154-1161.
Asunto(s)
Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/inmunología , Evasión Inmune , Neoplasias/patología , Animales , Línea Celular , Ganciclovir/farmacología , Técnicas de Sustitución del Gen , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Células Madre Embrionarias Humanas/efectos de los fármacos , Humanos , Evasión Inmune/efectos de los fármacos , Tolerancia Inmunológica/efectos de los fármacos , Ratones , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Factores de Riesgo , Teratoma/patologíaRESUMEN
Antibody fragments have shown targeted specificity to their antigens, but only modest tissue retention times in vivo and in vitro. Multimerization has been used as a protein engineering tool to increase the number of binding units and thereby enhance the efficacy and retention time of antibody fragments. In this work, we explored the effects of valency using a series of self-assembling polypeptides based on the GCN4 leucine zipper multimerization domain fused to a single-chain variable fragment via an antibody upper hinge sequence. Four engineered antibody fragments with a valency from one to four antigen-binding units of a cytotoxic monoclonal antibody 84 against human embryonic stem cells (hESC) were constructed. We hypothesized that higher cytotoxicity would be observed for fragments with increased valency. Flow cytometry analysis revealed that the trimeric and tetrameric engineered antibody fragments resulted in the highest degree of cytotoxicity to the undifferentiated hESC, while the engineered antibody fragments were observed to have improved tissue penetration into cell clusters. Thus, a trade off was made for the trimeric versus tetrameric fragment due to improved tissue penetration. These results have direct implications for antibody-mediated removal of undifferentiated hESC during regenerative medicine and cell therapy.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/toxicidad , Células Madre Embrionarias Humanas/efectos de los fármacos , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Afinidad de Anticuerpos , Antígenos/química , Cromatografía Líquida de Alta Presión , Pruebas Inmunológicas de Citotoxicidad , Escherichia coli/genética , Citometría de Flujo , Células Madre Embrionarias Humanas/inmunología , Humanos , Fragmentos de Inmunoglobulinas/genética , Plásmidos/genética , Multimerización de Proteína/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Análisis de Secuencia de ADN , Anticuerpos de Cadena Única/químicaRESUMEN
AIMS/HYPOTHESIS: To overcome the donor shortage in the treatment of advanced type 1 diabetes by islet transplantation, human embryonic stem cells (hESCs) show great potential as an unlimited alternative source of beta cells. hESCs may have immune privileged properties and it is important to determine whether these properties are preserved in hESC-derived cells. METHODS: We comprehensively investigated interactions of both innate and adaptive auto- and allo-immunity with hESC-derived pancreatic progenitor cells and hESC-derived endocrine cells, retrieved after in-vivo differentiation in capsules in the subcutis of mice. RESULTS: We found that hESC-derived pancreatic endodermal cells expressed relatively low levels of HLA endorsing protection from specific immune responses. HLA was upregulated when exposed to IFNγ, making these endocrine progenitor cells vulnerable to cytotoxic T cells and alloreactive antibodies. In vivo-differentiated endocrine cells were protected from complement, but expressed more HLA and were targets for alloreactive antibody-dependent cellular cytotoxicity and alloreactive cytotoxic T cells. After HLA compatibility was provided by transduction with HLA-A2, preproinsulin-specific T cells killed insulin-producing cells. CONCLUSIONS/INTERPRETATION: hESC-derived pancreatic progenitors are hypoimmunogenic, while in vivo-differentiated endocrine cells represent mature targets for adaptive immune responses. Our data support the need for immune intervention in transplantation of hESC-derived pancreatic progenitors. Cell-impermeable macro-encapsulation may suffice.
Asunto(s)
Células Madre Embrionarias Humanas/inmunología , Células Secretoras de Insulina/inmunología , Células Madre/metabolismo , Inmunidad Adaptativa/inmunología , Aloinjertos , Autoinmunidad , Células Cultivadas , Antígeno HLA-A2 , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Inmunidad Humoral/inmunología , Inmunidad Innata/inmunología , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Interferón gamma/metabolismoRESUMEN
Embryonic carcinoma (EC) cells are malignant counterparts of embryonic stem (ES) cells and serve as useful models for investigating cellular differentiation and human embryogenesis. Though the susceptibility of murine EC cells to retroviral infection has been extensively analyzed, few studies of retrovirus infection of human EC cells have been performed. We tested the susceptibility of human EC cells to transduction by retroviral vectors derived from three different retroviral genera. We show that human EC cells efficiently express reporter genes delivered by vectors based on human immunodeficiency virus type 1 (HIV-1) and Mason-Pfizer monkey virus (M-PMV) but not Moloney murine leukemia virus (MLV). In human EC cells, MLV integration occurs normally, but no viral gene expression is observed. The block to MLV expression of MLV genomes is relieved upon cellular differentiation. The lack of gene expression is correlated with transcriptional silencing of the MLV promoter through the deposition of repressive histone marks as well as DNA methylation. Moreover, depletion of SETDB1, a histone methyltransferase, resulted in a loss of transcriptional silencing and upregulation of MLV gene expression. Finally, we provide evidence showing that the lack of MLV gene expression may be attributed in part to the lack of MLV enhancer function in human EC cells. IMPORTANCE: Human embryonic carcinoma (EC) cells are shown to restrict the expression of murine leukemia virus genomes but not retroviral genomes of the lentiviral or betaretroviral families. The block occurs at the level of transcription and is accompanied by the deposition of repressive histone marks and methylation of the integrated proviral DNA. The host machinery required for silencing in human EC cells is distinct from that in murine EC cell lines: the histone methyltransferase SETDB1 is required, but the widely utilized corepressor TRIM28/Kap1 is not. A transcriptional enhancer element from the Mason-Pfizer monkey virus can override the silencing and promote transcription of chimeric proviral DNAs. The findings reveal novel features of human EC gene regulation not present in their murine counterparts.
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Silenciador del Gen , Genoma Viral , VIH-1/genética , Células Madre Embrionarias Humanas/inmunología , Virus del Mono Mason-Pfizer/genética , Virus de la Leucemia Murina de Moloney/genética , Células Madre Neoplásicas/inmunología , Animales , Diferenciación Celular , Metilación de ADN , Genes Reporteros , VIH-1/metabolismo , N-Metiltransferasa de Histona-Lisina , Histonas/genética , Histonas/inmunología , Especificidad del Huésped , Células Madre Embrionarias Humanas/virología , Humanos , Virus del Mono Mason-Pfizer/metabolismo , Ratones , Virus de la Leucemia Murina de Moloney/metabolismo , Células Madre Neoplásicas/virología , Regiones Promotoras Genéticas , Proteína Metiltransferasas/antagonistas & inhibidores , Proteína Metiltransferasas/genética , Proteína Metiltransferasas/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Especificidad de la Especie , Transcripción GenéticaRESUMEN
With ongoing clinical trials, human embryonic stem cells (hESCs) have shown substantial potential for regenerative medicine. However, due to the mismatch of human leukocyte antigens (HLAs) between hESC-derived allografts and recipients, immunosuppressant regimens must be used to prevent immune rejection of the grafts. Considerable efforts have been devoted to overcoming this hurdle via the derivation and banking of human nuclear transfer ESCs, parthenogenetic ESCs, and induced pluripotent stem cells. However, ethical and safety concerns remain, hindering the application of these types of pluripotent cells. Other approaches have recently been explored to generate universally compatible hESCs through the silencing or deletion of HLAs or genes essential for HLA expression, including ß-2-microglobulin and class-II MHC transactivator, as well as the induction of immunosuppression via the ectopic expression of non-classical HLAs (e.g., HLA-E and -G), cytotoxic T lymphocyte antigen 4 fused with immunoglobulin, and programmed death ligand-1. In this review, we introduce developments in this line of research and discuss strategies to reduce the tumorigenic concerns regarding hESCs, especially after they acquire the capability to escape immune surveillance. Stem Cells 2016;34:2269-2275.
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Técnicas de Cultivo de Célula/métodos , Células Madre Embrionarias Humanas/citología , Carcinogénesis/patología , Células Madre Embrionarias Humanas/inmunología , Humanos , Terapia de Inmunosupresión , Cordón Umbilical/citologíaRESUMEN
We have recently described sustained clinical recovery associated with dampened neuroinflammation and remyelination following transplantation of neural precursor cells (NPCs) derived from human embryonic stem cells (hESCs) in a viral model of the human demyelinating disease multiple sclerosis. The hNPCs used in that study were derived by a novel direct differentiation method (direct differentiation, DD-NPCs) that resulted in a unique gene expression pattern when compared to hNPCs derived by conventional methods. Since the therapeutic potential of human NPCs may differ greatly depending on the method of derivation and culture, we wanted to determine whether NPCs differentiated using conventional methods would be similarly effective in improving clinical outcome under neuroinflammatory demyelinating conditions. For the current study, we utilized hNPCs differentiated from a human induced pluripotent cell line via an embryoid body intermediate stage (EB-NPCs). Intraspinal transplantation of EB-NPCs into mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in decreased accumulation of CD4+ T cells in the central nervous system that was concomitant with reduced demyelination at the site of injection. Dampened neuroinflammation and remyelination was correlated with a transient increase in CD4+FOXP3+ regulatory T cells (Tregs) concentrated within the peripheral lymphatics. However, compared to our earlier study, pathological improvements were modest and did not result in significant clinical recovery. We conclude that the genetic signature of NPCs is critical to their effectiveness in this model of viral-induced neurologic disease. These comparisons will be useful for understanding what factors are critical for the sustained clinical improvement.
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Infecciones por Coronavirus/terapia , Cuerpos Embrioides/inmunología , Hepatitis Viral Animal/terapia , Células Madre Embrionarias Humanas/inmunología , Células-Madre Neurales/trasplante , Linfocitos T Reguladores/inmunología , Animales , Biomarcadores/metabolismo , Antígenos CD4/genética , Antígenos CD4/inmunología , Diferenciación Celular , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Modelos Animales de Enfermedad , Cuerpos Embrioides/citología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Expresión Génica , Hepatitis Viral Animal/inmunología , Hepatitis Viral Animal/patología , Hepatitis Viral Animal/virología , Células Madre Embrionarias Humanas/citología , Humanos , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Esclerosis Múltiple/terapia , Virus de la Hepatitis Murina/crecimiento & desarrollo , Virus de la Hepatitis Murina/patogenicidad , Vaina de Mielina/inmunología , Células-Madre Neurales/citología , Células-Madre Neurales/inmunología , Especificidad de Órganos , Linfocitos T Reguladores/patologíaRESUMEN
Human embryonic stem cells (hESCs) are thought to be a promising resource for cell therapy, while it has to face the major problem of graft immunological rejection. Major histocompatibility complex (MHC) class I expressed on the cell surface is the major cause of graft rejection. Transporter associated with antigen presentation 1 (TAP1) and TAP-associated glycoprotein (TAPBP) play important roles in regulating MHC class I expression. In this study, we generated TAP1- and TAPBP-deficient hESC lines, respectively, using transcription activator-like effector nucleases technique. These cells showed deficient expression of MHC class I on the cell surface and reduced immunogenicity compared with wild types, but maintained normal pluripotency, karyotypes, and differentiation ability. Thus, our findings are instrumental in developing a universal cell resource with both pluripotency and hypo-immunogenicity for transplantation therapy in the future.